The use of tobacco products, especially in combination with alcohol, has been associated with an increased incidence of cancer of the oral cavity. In this proposal, we will investigate a novel approach to carcinogenesis from tobacco-derived components by determining if genotoxic agents found in tobacco products cause formation of covalently closed circular extrachromosomal (episomal) DNA in human cells from the oral cavity. Our data, and those of other laboratories, indicate that episomes from human cells can contain protooncogenes, growth factor genes, and drug resistance genes. It is our hypothesis that cells with growth factor receptor genes, protooncogenes, or drug resistance genes on episomal DNA will have a selective growth advantage and hence accelerate tumor formation. In bacteria, antibiotic resistance can develop as a result of gene amplification on extrachromosomal (plasmid) DNA. Because they are located on plasmids, drug resistance genes are present in much higher copy number than they could be if they were located on bacterial chromosomes. In mammalian cells, the presence of c-myc, and a drug resistance gene on episomes has been established in two different human tumor cell lines. The epidermal growth factor receptor has been reported on episomal DNA in cells obtained from hamster epithelium after polycyclic aromatic hydrocarbon exposure. Formation of episomes can facilitate gene amplification in cells containing these structures. In this proposal, we will determine the role of tobacco products in the presence and absence of ethanol on formation of episomal DNA in established cell lines obtained from carcinomas of the head and neck, as well as in primary cultures of cells from patients who undergo surgical removal of tumors of the head and neck. To determine if cells containing episomes have a selective growth advantage over cells without episomes, colonies of cells (from cell lines exposed to carcinogens or tumor cells) will be recovered after growth in soft agar and the presence of episomes as well as gene amplification will be compared to levels of episomal DNA and gene amplification present in the initial population of cells. The detection of cells containing episomal DNA offers an opportunity to intervene in this sequence of events by elimination of cells containing genes that provide a selective growth advantage.